vol. 21 weekly issue 49 08 december 2016 - …. 21 | weekly issue 49 | 08 december 2016 ... exposed...
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www.eurosurveillance.org
Vol. 21 | Weekly issue 49 | 08 December 2016
E u r o p e ’ s j o u r n a l o n i n f e c t i o u s d i s e a s e e p i d e m i o l o g y, p r e v e n t i o n a n d c o n t r o l
Rapid communications
Highly pathogenic avian influenza A(H5N8) outbreaks: protection and management of exposed people in Europe, 2014/15 and 2016 2by C Adlhoch, IH Brown, SG Angelova, Á Bálint, R Bouwstra, S Buda, MR Castrucci, G Dabrera, Á Dán, C Grund, T Harder, W van der Hoek, K Krisztalovics, F Parry-Ford, R Popescu, A Wallensten, A Zdravkova, S Zohari, S Tsolova, P Penttinen
Research Articles
Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014 9by G Van der Auwera, A Bart, C Chicharro, S Cortes, L Davidsson, T Di Muccio, J Dujardin, I Felger, MG Paglia, F Grimm, G Harms, CL Jaffe, M Manser, C Ravel, F Robert-Gangneux, J Roelfsema, S Töz, JJ Verweij, PL Chiodini
Letters
Community-wide outbreaks of haemolytic uraemic syndrome associated with Shiga toxin-producing Escherichia coli O26 in Italy and Romania: a new challenge for the European Union 20by E Severi, F Vial, E Peron, O Mardh, T Niskanen, J Takkinen
News
New version of the Epidemic Intelligence Information System for food- and waterborne diseases and zoonoses (EPIS-FWD) launched 22by CM Gossner
2 www.eurosurveillance.org
Rapid communications
Highly pathogenic avian influenza A(H5N8) outbreaks: protection and management of exposed people in Europe, 2014/15 and 2016
C Adlhoch ¹ , IH Brown ² , SG Angelova ³ , Á Bálint ⁴ , R Bouwstra ⁵ , S Buda ⁶ , MR Castrucci ⁷ , G Dabrera ⁸ , Á Dán ⁴ , C Grund ⁹ , T Harder ⁹ , W van der Hoek 10 , K Krisztalovics 11 , F Parry-Ford ⁸ , R Popescu 12 , A Wallensten 13 , A Zdravkova 14 , S Zohari 15 , S Tsolova ¹ , P Penttinen ¹ 1. European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden2. Animal and Plant Health Agency (APHA), Weybridge, United Kingdom3. National Centre of Infectious and Parasitic Diseases, Sofia, Bulgaria4. National Food Chain Safety Office (NEBIH), Budapest, Hungary5. GD Animal Health Deventer, Netherlands6. Robert Koch Institute (RKI), Berlin, Germany7. Istituto Superiore di Sanita (ISS), Rome, Italy8. Public Health England (PHE), London, United Kingdom9. Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany10. National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands11. National Center for Epidemiology, Budapest, Hungary12. National Institute of Public Health, Bucharest, Romania13. The Public Health Agency of Sweden, Stockholm, Sweden14. Bulgarian Food Safety Agency, Sofia, Bulgaria15. National Veterinary Institute (SVA), Uppsala, SwedenCorrespondence: Cornelia Adlhoch ([email protected])
Citation style for this article: Adlhoch C, Brown IH, Angelova SG, Bálint Á, Bouwstra R, Buda S, Castrucci MR, Dabrera G, Dán Á, Grund C, Harder T, van der Hoek W, Krisztalovics K, Parry-Ford F, Popescu R, Wallensten A, Zdravkova A, Zohari S, Tsolova S, Penttinen P. Highly pathogenic avian influenza A(H5N8) outbreaks: protection and management of exposed people in Europe, 2014/15 and 2016. Euro Surveill. 2016;21(49):pii=30419. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2016.21.49.30419
Article submitted on 29 November 2016 / accepted on 07 December 2016 / published 08 December 2016
Introduction of highly pathogenic avian influenza (HPAI) virus A(H5N8) into Europe prompted animal and human health experts to implement protective meas-ures to prevent transmission to humans. We describe the situation in 2016 and list public health measures and recommendations in place. We summarise critical interfaces identified during the A(H5N1) and A(H5N8) outbreaks in 2014/15. Rapid exchange of information between the animal and human health sectors is criti-cal for a timely, effective and efficient response.
Avian influenza A(H5N8) situation in Europe, December 2016In September 2016, the Food and Agriculture Organization (FAO) of the United Nations raised aware-ness for the potential reintroduction of highly patho-genic avian influenza (HPAI) virus A(H5N8) to Europe after the detection in a wild swan in the Tyva Republic, Russia, in June 2016. A potential spread of the virus was assumed via the migratory bird routes of duck, geese and swans [1]. The communication followed ear-lier reports in 2016, of A(H5N8) in wild and domestic birds in the Republic of Korea, and Taiwan, and the event suggested re-introduction of the virus via wild birds migrating back to Europe for overwintering.
Outbreaks in wild birdsFrom 30 October to 6 December 2016, 14 European countries (Austria, Croatia, Denmark, Finland, France, Germany, Hungary, the Netherlands, Poland, Romania, Russia, Serbia, Switzerland, and Sweden) as well as Egypt, India, Iran, and Israel reported HPAI A(H5N8) outbreaks in domestic poultry or detections in wild or zoo birds (Figure) [2]. Tunisia and Ukraine reported HPAI A(H5) outbreaks suspected to be A(H5N8). Since the first finding in October, the virus spread rapidly across central Europe. It mostly affected wild water birds, but also birds of prey that feed on dead birds’ carcasses. Infections of the latter indicate a recent introduction into the local resident bird population.
Outbreaks in poultry holdingsIn 2016, outbreaks in poultry holdings were reported from Austria, Denmark, France, Germany, Hungary, the Netherlands, Poland and Sweden [2]. This resem-bles the situation in the northern hemisphere winter 2014/15 when a virus of the same clade 2.3.4.4 caused outbreaks in six European countries (Germany [3,4], Italy [5], Hungary [6], the Netherlands [7], Sweden and the United Kingdom [8]), mainly in closed poultry holdings, and sporadic detections in wild birds and a zoo [2,3]. Although the viruses belong to the same
3www.eurosurveillance.org
genotype clade, viruses during the 2014/15 outbreak belonged to a different group of clade 2.3.4.4, group A (Buan-like), while the current 2016 viruses cluster in clade 2.3.4.4 group B (Gochang-like).
This report presents critical points identified during the HPAI A(H5N1) and A(H5N8) outbreaks in 2014/15 for preparedness, communication and public as well as animal health recommendations and measures to con-tain outbreak of avian influenza.
Potential risks to human healthNo human cases of influenza A(H5N8) virus infection have been reported despite large numbers of people being occupationally exposed while managing the avian outbreaks, thus the risk for humans is consid-ered very low [9]. This contrasts with the risk of bird-to-human transmission of influenza A(H5N1) and is likely due to A(H5N8) receptor-binding properties with the latter virus being better adapted to avian-like recep-tors than human-like receptors [8,10-12]. Although the
sequence information available for the haemagglutinin and neuraminidase proteins of recent A(H5N8) iso-lates does not show any evolution towards increased affinity for humans, these viruses should be closely monitored for any adaptation [13]. The non-structural protein (NS) gene of the A(H5N8) virus detected in a wild sea duck, common Goldeneye, in Sweden in mid-November is truncated (217aa) and reassortment in polymerase acidic (PA) and nucleoprotein (NP) genes has been observed compared to those viruses detected earlier in June in Tyva (S. Zohari, personal communication, December 2016; sequences available in GISAID: EPI863862-69; National Veterinary Institute; Uppsala, Sweden A/Common Goldeneye/Sweden/SVA161117KU0322/SZ0002165/2016).
Influenza viruses undergo constant reassortment. Recent human cases of influenza A(H5N6) reported from China illustrate how A(H5) viruses belonging to the same clade 2.3.4.4 as A(H5N8) viruses, can gain the ability to infect humans without any of the major
Figure Detection of highly pathogenic avian influenza virus A(H5N8) in wild birds and poultry, Europe and neighbouring regions, November 2014 to February 2015 and October to December 2016a
2014/1520162016 suspicion
EU/EEA: European Union/European Economic Area.
a The map displays the situation as at 6 December 2016. Dark grey represents the EU/EEA.
4 www.eurosurveillance.org
Tabl
e a
Avia
n in
fluen
za p
reve
ntio
n an
d co
ntro
l mea
sure
s im
plem
ente
d by
sele
cted
nat
iona
l Eur
opea
n U
nion
pub
lic h
ealth
aut
horit
iesa , D
ecem
ber 2
016
Mea
sure
B
ulga
ria
Engl
and
Germ
any
aHu
ngar
yIta
ly b
Net
herla
nds
cRo
man
iaSw
eden
Prot
ectio
n of
ex
pose
d in
divi
dual
s du
ring
avi
an
infl
uenz
a in
cide
nts
Indi
vidu
als
expo
sed
occu
patio
nally
or i
n cl
ose
cont
act w
ith in
fect
ed
or p
oten
tially
infe
cted
bi
rds
shou
ld u
se s
uita
ble
PPE
such
as
disp
osab
le
over
alls
, nitr
ile/v
inyl
gl
oves
, rub
ber b
oots
, go
ggle
s, a
nd a
filte
ring
ha
lf m
ask
with
exh
alat
ion
valv
e. T
he p
erso
nnel
ha
ndlin
g in
fect
ed o
r po
tent
ially
infe
cted
bi
rds
shou
ld o
bser
ve th
e bi
osec
urit
y in
stru
ctio
ns
for c
olle
ctio
n an
d di
spos
al o
f the
equ
ipm
ent
used
.
Indi
vidu
als
expo
sed
occu
patio
nally
sho
uld
use
appr
opri
ate
PPE:
di
spos
able
or p
olyc
otto
n ov
eral
l, di
spos
able
gl
oves
, rub
ber o
r po
lyur
etha
ne b
oots
, FF
P3 re
spir
ator
with
ex
hala
tion
valv
e an
d cl
ose
fittin
g go
ggle
s.
PPE
incl
udin
g di
spos
able
glo
ves,
cl
othi
ng, h
eadw
ear,
prot
ectiv
e bo
ots,
cl
ose
fittin
g go
ggle
s an
d m
asks
: FFP
1 if
aero
solis
atio
n is
not
lik
ely,
oth
erw
ise
FFP3
w
ith e
xhal
atio
n va
lve
Full
body
pro
tect
ion:
(ove
rall,
gl
oves
, boo
ts, g
oggl
es) a
nd F
FP3
resp
irat
or
Occ
upat
iona
lly
expo
sed
indi
vidu
als
shou
ld
use
appr
opri
ate
PPE:
FFP
3 m
asks
, ru
bber
glo
ves
and
boot
s re
sist
ant
to d
eter
gent
s/di
sinf
ecta
nts,
di
spos
able
ove
rall
and
hair
cove
r, ey
e pr
otec
tion.
Eye
prot
ectio
n, F
FP2
mas
k (fo
r cul
lers
FFP
3),
boot
s, d
ispo
sabl
e ov
eral
l, ha
ir co
ver,
disp
osab
le g
love
s
Mea
sure
s fo
r pr
otec
ting
the
indi
vidu
als
who
co
me
in c
onta
ct w
ith
infe
cted
bird
s or
like
ly
to b
e in
fect
ed, b
irds
aliv
e or
dea
d ar
e:
PPE
and
appr
opri
ate
cond
ition
s fo
r co
llect
ion,
ne
utra
lisat
ion
and
stor
age
of th
e eq
uipm
ent u
sed.
Indi
vidu
als
occu
patio
nally
ex
pose
d sh
ould
us
e ap
prop
riat
e PP
E: d
ispo
sabl
e or
pol
ycot
ton
over
all,
disp
osab
le
glov
es, r
ubbe
r or
poly
uret
hane
boo
ts,
FFP3
resp
irat
or w
ith
exha
latio
n va
lve
and
clos
e fit
ting
gogg
les.
Peri
od th
at e
xpos
ed
peop
le s
houl
d be
mon
itore
d fo
r sy
mpt
oms
7-10
day
s10
day
s7
days
10 d
ays
Up to
10
days
fo
llow
ing
expo
sure
Poul
try
wor
kers
/
culle
rs a
re re
ques
ted
to
repo
rt s
ympt
oms
until
10
day
s po
st e
xpos
ure,
to
the
mun
icip
al h
ealth
se
rvic
e, i.
e. p
assi
ve
mon
itori
ng, w
hich
can
be
sca
led
up to
act
ive
mon
itori
ng.
7 da
ys10
day
s
Test
ing
Clin
ical
spe
cim
ens
(nas
o-ph
aryn
geal
sw
abs/
br
onch
oalv
eola
r lav
age
fluid
/end
otra
chea
l as
pira
te/
pleu
ral f
luid
/ sp
utum
) fro
m e
xpos
ed
peop
le w
ith re
spir
ator
y sy
mpt
oms
in c
lose
co
ntac
t with
ill o
r dea
d bi
rds,
thei
r fam
ily
mem
bers
or t
rave
llers
to
coun
trie
s w
ith re
gist
ered
av
ian
influ
enza
cas
es w
ill
be c
olle
cted
to id
entif
y in
fluen
za a
nd s
peci
fical
ly
A(H5
).
Influ
enza
and
sp
ecifi
cally
A(H
5)
from
resp
irat
ory
trac
t sa
mpl
es
Influ
enza
and
sp
ecifi
cally
A(H
5)
from
resp
irat
ory
trac
t sa
mpl
es;
if se
rolo
gica
l tes
ting
is c
onsi
dere
d pa
ired,
se
rum
sam
ples
sho
uld
be c
olle
cted
.
Test
for h
uman
influ
enza
A,B
,C
and
influ
enza
A/H
5;
resp
irat
ory
trac
t and
pai
red
(10-
14 d
ays)
ser
um s
ampl
es s
houl
d be
take
n an
d se
nt to
refe
renc
e la
bora
tory
of N
CE.
Influ
enza
and
A/
H5 fr
om re
spir
ator
y tr
act s
ampl
es;
paire
d se
rum
sa
mpl
es s
houl
d al
so b
e ta
ken.
Test
ing
only
aft
er
tele
phon
e co
nsul
tatio
n w
ith th
e vi
rolo
gist
on
duty
(24/
7) a
t RIV
M;
nose
, thr
oat,
and
eye
swab
for P
CR a
naly
sis.
Naso
-pha
ryng
eal
swab
s w
ill b
e co
llect
ed to
iden
tify
avia
n in
fluen
za v
irus
A(
H5).
Influ
enza
and
sp
ecifi
cally
A(H
5)
from
resp
irat
ory
trac
t sam
ples
FFP:
filte
ring
face
pie
ce; N
A: n
ot a
pplic
able
; NCE
: Nat
iona
l Cen
tre
for E
pide
mio
logy
; RIV
M: N
atio
nal I
nstit
ute
for P
ublic
Hea
lth a
nd th
e En
viro
nmen
t; P
PE: p
erso
nal p
rote
ctiv
e eq
uipm
ent;
RIV
M: R
ijksi
nstit
uut v
oor V
olks
gezo
ndhe
id e
n M
ilieu
(The
Dut
ch N
atio
nal I
nstit
ute
for P
ublic
Hea
lth a
nd th
e En
viro
nmen
t); U
NKN:
unk
now
n; W
HO: W
orld
Hea
lth O
rgan
izat
ion.
a The
info
rmat
ion
prov
ided
app
lies
gene
rally
, but
eac
h in
cide
nt is
ass
esse
d in
divi
dual
ly to
ens
ure
a fu
lly a
ppro
pria
te re
spon
se.
b Nat
iona
l gui
delin
es in
Ital
y le
ave
flexi
bilit
y to
pro
vide
ant
ivir
al p
roph
ylax
is d
urin
g av
ian
influ
enza
out
brea
ks, w
here
as v
acci
natio
n w
ith s
easo
nal v
acci
ne is
ann
ually
reco
mm
ende
d fo
r vet
erin
ary
serv
ice
and
poul
try/
swin
e in
dust
ry
wor
kers
.c N
atio
nal g
uide
lines
in th
e Ne
ther
land
s le
ave
cons
ider
able
flex
ibili
ty re
gard
ing
mon
itori
ng, a
ntiv
iral
pro
phyl
axis
and
sea
sona
l vac
cina
tion.
The
app
roac
h is
tailo
r-m
ade
to e
nsur
e a
rapi
d an
d fle
xibl
e re
spon
se to
any
sig
nal.
5www.eurosurveillance.org
Mea
sure
B
ulga
ria
Engl
and
Germ
any
aHu
ngar
yIta
ly b
Net
herla
nds
cRo
man
iaSw
eden
Pre
or p
ost-
expo
sure
ch
emop
roph
ylax
is
Expo
sed
indi
vidu
als:
ant
ivir
al
chem
opro
phyl
axis
, im
med
iate
ly a
fter
ex
posu
re
Indi
vidu
als
who
are
ex
pose
d oc
cupa
tiona
lly
may
be
offe
red
antiv
iral
ch
emop
roph
ylax
is a
s an
add
ed p
reca
utio
n fo
llow
ing
an a
ppro
pria
te
risk
ass
essm
ent a
nd
acco
rdin
g to
def
ined
al
gori
thm
.
Expo
sed
indi
vidu
als
with
dire
ct c
onta
ct to
in
fect
ed b
irds
follo
win
g an
app
ropr
iate
risk
as
sess
men
t (fo
r ex
ampl
e ap
prop
riat
e PP
E du
ring
exp
osur
e or
not
)
Post
-exp
osur
e pr
ophy
laxi
s:
osel
tam
ivir
antiv
iral
pro
phyl
axis
fo
r 10
days
for o
ccup
atio
nally
ex
pose
d in
divi
dual
s
Expo
sed
indi
vidu
als
on
eval
uatio
n by
loca
l he
alth
aut
hori
ties
All e
xpos
ed w
orke
rs,
farm
ers,
and
thei
r fa
mily
mem
bers
; a
natio
nal s
uppl
y of
an
tivir
als
is k
ept a
t RI
VM.
Spec
ific
mea
sure
s to
pro
tect
exp
osed
in
divi
dual
s:
prop
hyla
ctic
ant
ivir
al
trea
tmen
t for
7 d
ays,
im
med
iate
ly a
fter
ex
posu
re
Indi
vidu
als
who
hav
e be
en
expo
sed
with
out
wea
ring
pro
tect
ive
equi
pmen
t de
pend
ing
on
the
type
of a
vian
in
fluen
za a
nd th
e ex
posu
re
PPE
and
othe
r pr
ecau
tion
mea
sure
s to
be
used
by
heal
thca
re
wor
kers
ass
essi
ng
sym
ptom
atic
, ex
pose
d pe
ople
PPE:
dis
posa
ble
glov
es;
sing
le u
se m
outh
/nos
e m
ask,
gog
gles
; st
anda
rd, c
onta
ct a
nd
airb
orne
pre
caut
ions
Cont
act a
nd a
irbo
rne
prec
autio
ns; t
his
incl
udes
eye
pro
tect
ion,
FF
P3 re
spir
ator
, gow
ns
and
glov
es w
hen
wor
king
in s
ame
room
as
the
sym
ptom
atic
per
son.
Stan
dard
, con
tact
and
ai
rbor
ne p
reca
utio
ns;
eye
prot
ectio
n
Stan
dard
, con
tact
and
dro
plet
-ai
rbor
ne p
reca
utio
ns w
ith e
ye
prot
ectio
n
Stan
dard
, con
tact
an
d ai
rbor
ne
prec
autio
ns,
incl
udin
g ey
e pr
otec
tion
Stan
dard
PPE
and
pe
rson
al h
ygie
ne
mea
sure
s
PPE:
sin
gle
use
gow
ns, s
ingl
e us
e m
outh
/nos
e m
ask,
go
ggle
s, s
ingl
e us
e gl
oves
; sta
ndar
d co
ntac
t and
air
born
e pr
ecau
tions
Stan
dard
, con
tact
an
d ai
rbor
ne
prec
autio
ns;
eye
prot
ectio
n;
gow
ns a
nd g
love
s w
hen
wor
king
in
sam
e ro
om a
s th
e sy
mpt
omat
ic p
erso
n
Seas
onal
in
flue
nza
vacc
ine
reco
mm
enda
tion
Risk
pop
ulat
ion
grou
ps
reco
mm
ende
d by
WHO
for
influ
enza
vac
cina
tion
As p
er u
sual
ann
ual
reco
mm
enda
tions
for
at-r
isk
grou
ps
As p
er re
com
men
datio
n of
the
Ger
man
st
andi
ng c
omm
itte
e fo
r va
ccin
atio
n (S
TIKO
), in
clud
ing
poul
try
wor
kers
Base
d on
the
reco
mm
enda
tion
of N
CE (i
n th
e an
nual
circ
ular
of
the
Chie
f Med
ical
Off
icer
): po
ultr
y/pi
g w
orke
rs (b
reed
ers,
tr
ansp
orte
rs, c
ulle
rs, w
orke
rs in
pr
oces
sing
pla
nts,
etc
.)
Poul
try/
pig
wor
kers
an
d he
alth
care
w
orke
rs
Onl
y va
ccin
ated
wor
kers
ca
n be
invo
lved
in
culli
ng. V
acci
natio
n is
off
ered
to o
ther
w
orke
rs, f
arm
ers
and
thei
r fam
ily m
embe
rs if
ou
tbre
aks
occu
r dur
ing
influ
enza
sea
son.
Risk
pop
ulat
ion
grou
ps re
com
men
ded
by W
HO fo
r inf
luen
za
vacc
inat
ion
(incl
udin
g HC
W) a
nd fo
r ex
pose
d in
divi
dual
s,
in o
rder
to a
void
th
e re
asso
rtm
ent
betw
een
hum
an a
nd
avia
n vi
rus
As p
er u
sual
re
com
men
datio
ns,
curr
ently
no
requ
irem
ent f
or
poul
try
wor
kers
Mea
sure
s (a
pplie
d or
pla
nned
) to
follo
w-u
p on
ex
pose
d in
divi
dual
s du
ring
cur
rent
A(
H5N8
) out
brea
ks
or d
etec
tions
in
bird
s
NA
Expo
sed
indi
vidu
als
will
be
follo
wed
up
eith
er
activ
ely
or p
assi
vely
du
ring
inci
dent
and
fo
r 10
days
aft
er la
st
expo
sure
. Cho
ice
of a
ctiv
e or
pas
sive
fo
llow
-up
depe
nds
on ty
pe o
f exp
osur
e an
d an
ass
essm
ent
of u
se o
f pro
tect
ive
mea
sure
s, in
clud
ing
chem
opro
phyl
axis
if
indi
cate
d.
UNKN
Iden
tific
atio
n of
indi
vidu
als
expo
sed
and
/ or
invo
lved
in th
e cu
lling
; ind
ivid
uals
with
spe
cific
co
nditi
ons
(und
erly
ing
dise
ases
, im
mun
odef
icie
ncie
s, p
regn
ancy
, in
divi
dual
s ol
der t
han
62 y
ears
of
age
) sho
uld
be e
xclu
ded
from
cu
lling
. In
form
atio
n of
exp
osed
in
divi
dual
s ab
out t
he ri
sk, t
he
sign
s an
d sy
mpt
oms
of th
e di
seas
e an
d th
e m
etho
ds o
f pr
even
tion.
As
sign
ing
a ph
ysic
ian
resp
onsi
ble
for m
onito
ring
the
heal
th o
f the
exp
osed
for 1
0 da
ys.
NA
UNKN
UNKN
Pass
ive
surv
eilla
nce
of th
ose
expo
sed
with
out p
rope
r pr
otec
tive
equi
pmen
t
Tabl
e b
Avia
n in
fluen
za p
reve
ntio
n an
d co
ntro
l mea
sure
s im
plem
ente
d by
sele
cted
nat
iona
l Eur
opea
n U
nion
pub
lic h
ealth
aut
horit
iesa , D
ecem
ber 2
016
FFP:
filte
ring
face
pie
ce; N
A: n
ot a
pplic
able
; NCE
: Nat
iona
l Cen
tre
for E
pide
mio
logy
; RIV
M: N
atio
nal I
nstit
ute
for P
ublic
Hea
lth a
nd th
e En
viro
nmen
t; P
PE: p
erso
nal p
rote
ctiv
e eq
uipm
ent;
RIV
M: R
ijksi
nstit
uut v
oor V
olks
gezo
ndhe
id e
n M
ilieu
(The
Dut
ch N
atio
nal I
nstit
ute
for P
ublic
Hea
lth a
nd th
e En
viro
nmen
t); U
NKN:
unk
now
n; W
HO: W
orld
Hea
lth O
rgan
izat
ion.
a The
info
rmat
ion
prov
ided
app
lies
gene
rally
, but
eac
h in
cide
nt is
ass
esse
d in
divi
dual
ly to
ens
ure
a fu
lly a
ppro
pria
te re
spon
se.
b Nat
iona
l gui
delin
es in
Ital
y le
ave
flexi
bilit
y to
pro
vide
ant
ivir
al p
roph
ylax
is d
urin
g av
ian
influ
enza
out
brea
ks, w
here
as v
acci
natio
n w
ith s
easo
nal v
acci
ne is
ann
ually
reco
mm
ende
d fo
r vet
erin
ary
serv
ice
and
poul
try/
swin
e in
dust
ry
wor
kers
.c N
atio
nal g
uide
lines
in th
e Ne
ther
land
s le
ave
cons
ider
able
flex
ibili
ty re
gard
ing
mon
itori
ng, a
ntiv
iral
pro
phyl
axis
and
sea
sona
l vac
cina
tion.
The
app
roac
h is
tailo
r-m
ade
to e
nsur
e a
rapi
d an
d fle
xibl
e re
spon
se to
any
sig
nal.
6 www.eurosurveillance.org
Mea
sure
B
ulga
ria
Engl
and
Germ
any
aHu
ngar
yIta
ly b
Net
herla
nds
cRo
man
iaSw
eden
Sero
epid
emio
logi
cal
follo
w-u
p pl
anne
d in
20
16/1
7 No
Not r
outin
ely
done
; foc
us
is o
n in
vest
igat
ion
of
sym
ptom
atic
indi
vidu
al
patie
nts
expo
sed
duri
ng
inci
dent
s.
NoNo
No
In c
ase
of a
n ou
tbre
ak
ther
e is
a re
sear
ch
prot
ocol
whi
ch
incl
udes
ser
olog
y at
T0
and
T4w
eeks
– w
ith
anal
ysis
for d
iffer
ent
hum
an, a
vian
, sw
ine
influ
enza
vir
uses
usi
ng
mic
roar
ray.
NoNo
Oth
er s
tudi
es
plan
ned
rela
ted
to
curr
ent o
utbr
eaks
No
Will
be
dete
rmin
ed o
n an
inci
dent
-by-
inci
dent
ba
sis,
if re
quire
d to
su
ppor
t the
pub
lic
heal
th re
spon
se
UNKN
In o
rder
to a
sses
s th
e ef
ficac
y of
di
sinf
ectio
n, 1
00 g
dus
t sam
ples
ar
e ta
ken.
Wet
ted
tissu
e sw
abs
are
appl
ied
on 9
00 c
m2 s
urfa
ce
from
diff
eren
t par
ts o
f the
pen
re
pres
entin
g th
e w
hole
are
a.
No
Poss
ible
air
sam
plin
g in
and
aro
und
poul
try
farm
s in
cas
e of
a n
ew
outb
reak
NoNo
FFP:
filte
ring
face
pie
ce; N
A: n
ot a
pplic
able
; NCE
: Nat
iona
l Cen
tre
for E
pide
mio
logy
; RIV
M: N
atio
nal I
nstit
ute
for P
ublic
Hea
lth a
nd th
e En
viro
nmen
t; P
PE: p
erso
nal p
rote
ctiv
e eq
uipm
ent;
RIV
M: R
ijksi
nstit
uut v
oor V
olks
gezo
ndhe
id e
n M
ilieu
(The
Dut
ch N
atio
nal I
nstit
ute
for P
ublic
Hea
lth a
nd th
e En
viro
nmen
t); U
NKN:
unk
now
n; W
HO: W
orld
Hea
lth O
rgan
izat
ion.
a The
info
rmat
ion
prov
ided
app
lies
gene
rally
, but
eac
h in
cide
nt is
ass
esse
d in
divi
dual
ly to
ens
ure
a fu
lly a
ppro
pria
te re
spon
se.
b Nat
iona
l gui
delin
es in
Ital
y le
ave
flexi
bilit
y to
pro
vide
ant
ivira
l pro
phyl
axis
dur
ing
avia
n in
fluen
za o
utbr
eaks
, whe
reas
vac
cina
tion
with
sea
sona
l vac
cine
is a
nnua
lly re
com
men
ded
for v
eter
inar
y se
rvic
e an
d po
ultr
y/sw
ine
indu
stry
wor
kers
.c N
atio
nal g
uide
lines
in th
e Ne
ther
land
s le
ave
cons
ider
able
flex
ibili
ty re
gard
ing
mon
itorin
g, a
ntiv
iral p
roph
ylax
is a
nd s
easo
nal v
acci
natio
n. T
he a
ppro
ach
is ta
ilor-
mad
e to
ens
ure
a ra
pid
and
flexi
ble
resp
onse
to a
ny s
igna
l.
Tabl
e c
Avia
n in
fluen
za p
reve
ntio
n an
d co
ntro
l mea
sure
s im
plem
ente
d by
sele
cted
nat
iona
l Eur
opea
n U
nion
pub
lic h
ealth
aut
horit
iesa , D
ecem
ber 2
016
7www.eurosurveillance.org
adaptation processes referred to above. The current properties of the virus are not suggestive of pandemic risk. Still, the likely lack of immunity in humans against A(H5N8) and its increasing geographic distribution and incidence in animals justify constant monitoring of out-breaks in birds. Current concerns among veterinarians include the potential ability of A(H5N8) to infect mam-mals such as cats and dogs: thus precautions should be put in place to minimise the risk of exposure for these animals.
Available guidance on protective measuresAlthough the risk of human infection is considered very low [14], most of the available national guidance documents recommend a number of risk mitigation measures to minimise exposure. They target different groups: (i) for the general population recommenda-tions are to avoid exposure to potentially infected birds by not touching dead wild birds, and instead inform local veterinary authorities; (ii) local public and veteri-nary health authorities are recommended to limit the number of individuals exposed to birds suspected or confirmed to have HPAI; and (iii) individuals exposed occupationally are recommended to use appropriate personal protective equipment (PPE).
Experiences from 2014/15 outbreaks and measures in 2016In October 2015, animal and public health experts involved in the HPAI A(H5N1) and A(H5N8) outbreaks in Europe in 2014/15, reviewed relevant national proto-cols available in European Union/European Economic Area (EU/EEA) countries, actions implemented and les-sons learnt, in a workshop organised by the European Centre for Disease Prevention and Control (ECDC) [15].
The Table summarises key recommendations from selected public health authorities that managed avian influenza outbreaks in recent years and are valid in 2016. Generally they contain strict use of PPE when handling potentially infected birds, carcasses or other material, with some flexibility based on the local risk assessment in some countries. Post-exposure prophy-laxis with neuraminidase inhibitors is advised, often based on individual clinical assessment or local risk assessment. Some, but not all countries recommend pre-exposure prophylaxis that is to be continued during and after exposure. All countries recommend follow-up, passive or active, of those exposed, for development of symptoms for the duration of the maximum incubation period estimated to be around 7-10 days. Exposed peo-ple with influenza-like symptoms according to the EU case definition [16] should immediately be tested for influenza virus infection, preferably using lower respir-atory tract specimens and including H5-specific tests. Healthcare workers managing suspect human cases should take appropriate contact and airborne precau-tion measures (Table).
The experts concluded that the actions taken dur-ing the 2014/15 outbreaks were adequate to prevent
human cases, but some challenges and discrepancies were noted. There was agreement that timely sharing of information between the animal and human health sectors as well as between countries is crucial for an appropriate and early response. Intersectoral commu-nication should also continue between outbreaks to foster cooperation at national level. Most countries appear to use a maximum level of precaution during incidents rather than basing precautions on a careful risk assessment, and experts concur whether this was the most efficient approach. Although a general over-view of published evidence was considered useful, risk should be assessed locally.
Recommendations on use of antivirals or seasonal vac-cines differed between countries. Some challenges were encountered when post-exposure prophylaxis was recommended, but sufficient antivirals were not immediately available. The experts suggested that rapid availability of antivirals in each country should be reviewed and ensured.
Recommendations for seasonal influenza vaccination of poultry workers in general differ between countries. Seasonal influenza vaccination of exposed individuals during an outbreak was suggested in most countries to avoid co-infection with seasonal and avian influ-enza viruses which could be followed by reassortment events. However, England considered vaccination with seasonal influenza vaccine during an avian influenza outbreak as being too late for exposed individuals to develop an antibody response necessary for individual protection.
Active follow-up of exposed individuals is resource-demanding and requires a risk assessment. Suggestions from the meeting were to develop a tool to track and trace information on detections and out-breaks in animals as well as related human exposure and follow-up measures, in real time.
Streamlined messages based on evidence and targeted to those concerned are necessary. Communication bar-riers i.e. language were identified as reason for failure to follow up exposed mobile and migrant workers on poultry farms. This could be remedied by providing leaflets in different languages.
Large farms might have better safety and training standards than small farms, but response capacity and timeliness during outbreaks may still be insufficient.
Rapid communication and sharing of the viral genetic information is important to estimate the reliability of the PCR-based A(H5) HA gene detection applied in each country/region and antiviral treatment efficacy.
ConclusionsHumans have been and will be exposed to influenza A(H5N8) virus from infected birds, their carcasses or contaminated material in the coming weeks in Europe.
8 www.eurosurveillance.org
Although no human cases of influenza A(H5N8) have been documented, expert advice is that precautionary measures should be taken to minimise human expo-sure and possible infections. Relevant guidance and protection measures have proven sufficient during the avian influenza outbreaks in 2014/15 but were critically reviewed and adjusted where necessary. Well-designed follow-up studies among the exposed would help to document the (lack of) risk from A(H5N8) to humans and the effectiveness of control measures.
Timely communication between the animal and human health sectors is vital to enable a rapid, effective and efficient response to the ongoing outbreaks. Any human infection with a novel influenza subtype should trigger an immediate international notification through the International Health Regulations (IHR) mechanism and the EU Early Warning and Response System.
AcknowledgementsWe acknowledge the authors, originating and submit-ting laboratories of the sequences from GISAID’s EpiFlu Database. The submitter of data may be contacted directly via the GISAID website www.gisaid.org.
The authors are grateful to Kaja Kaasik Aaslav for her great support in monitoring avian influenza situation.
Conflict of interestNone declared.
Authors’ contributionsAll authors provided information on public health measures in their respective country, participated in the meeting re-ferred to in the article, contributed to the article and ap-proved the final version.
Cornelia Adlhoch coordinated the work, interpreted the data and led the writing of the article.
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License and copyrightThis is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi-cate if changes were made.
This article is copyright of the authors, 2016.
9www.eurosurveillance.org
Research article
Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014
G Van der Auwera ¹ , A Bart ² , C Chicharro ³ , S Cortes ⁴ , L Davidsson ⁵ , T Di Muccio ⁶ , J Dujardin 1 7 , I Felger 8 9 , MG Paglia 10 , F Grimm 11 , G Harms 12 , CL Jaffe 13 , M Manser 14 , C Ravel 15 , F Robert-Gangneux 16 , J Roelfsema 17 , S Töz 18 , JJ Verweij 19 , PL Chiodini 20 21 1. Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium2. Academic Medical Center, Amsterdam, The Netherlands3. Instituto de Salud Carlos III, Madrid, Spain4. Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, UNL, Lisbon, Portugal5. The Public Health Agency of Sweden, Stockholm, Sweden6. Istituto Superiore di Sanità, Rome, Italy7. Biomedical Sciences, Antwerp University, Antwerp, Belgium8. Swiss Tropical and Public Health Institute, Basel, Switzerland9. University of Basel, Basel, Switzerland10. National Institute for Infectious Diseases (INMI) Lazzaro Spallanzani, Rome, Italy11. Institute of Parasitology, University of Zürich, Zürich, Switzerland12. Institute of Tropical Medicine and International Health, Charité-Universitätsmedizin Berlin, Berlin, Germany13. Hebrew University, Hadassah Medical Centre, Jerusalem, Israel14. United Kingdom National External Quality Assessment Service, London, United Kingdom15. University of Montpellier, Montpellier, France16. Centre Hospitalier Universitaire de Rennes, Rennes, France17. National Institute for Public Health and the Environment, RIVM, Bilthoven, The Netherlands18. Ege University, Faculty of Medicine, Department of Parasitology, Izmir, Turkey19. St. Elisabeth Hospital, Tilburg, The Netherlands20. Hospital for Tropical Diseases, London, United Kingdom21. London School of Hygiene and Tropical Medicine, London, United KingdomCorrespondence: Gert Van der Auwera ([email protected])
Citation style for this article: Van der Auwera G, Bart A, Chicharro C, Cortes S, Davidsson L, Di Muccio T, Dujardin J, Felger I, Paglia MG, Grimm F, Harms G, Jaffe CL, Manser M, Ravel C, Robert-Gangneux F, Roelfsema J, Töz S, Verweij JJ, Chiodini PL. Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014. Euro Surveill. 2016;21(49):pii=30418. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2016.21.49.30418
Article submitted on 08 January 2016 / accepted on 13 July 2016 / published on 08 December 2016
Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accu-rate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were iden-tified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, inter-pretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array
should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.
IntroductionLeishmaniasis is a vector-borne disease which is endemic in 98 countries worldwide [1]. It is caused by protozoan parasites of the genus Leishmania, which are transmitted by female sand flies of the genera Lutzomyia and Phlebotomus. Many infected individuals never develop symptoms, but those who do can exhibit various disease manifestations [2]. Visceral leishma-niasis (VL) or kala-azar is the severe form, whereby parasites infect internal organs and the bone marrow, a lethal condition if left untreated. Other disease types are restricted to the skin (cutaneous leishmaniasis, CL) or the mucosae of the nose and mouth (mucosal leishmaniasis, ML). Finally, a particular cutaneous dis-ease sometimes develops in cured VL patients: post kala-azar dermal leishmaniasis (PKDL). Typically, VL is caused by two species: Leishmania donovani and Leishmania infantum. The latter can also cause CL, as can all other pathogenic species. Some particular
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species (e.g. L. braziliensis and L. aethiopica) can lead to overt ML.
As many as 20 different Leishmania species are able to infect humans, and globally there are over 1 million new disease cases per annum [1,3]. Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Although treatment in practice is often guided only by clinical presentation and patient his-tory, in some cases determination of the aetiological
subgenus, species complex or species is recommended for providing optimal treatment [2,4,5]. For exam-ple, a patient returning from South America with CL might be infected with Leishmania braziliensis, which necessitates systemic drug therapy and counselling about the risk of developing mucosal leishmaniasis in the future. The same patient could also be infected with Leishmania mexicana, which is managed by less intensive treatment and which is not associated with mucosal disease [6]. Determining the infecting spe-cies and its probable source permits selection of the
Figure 1Typing results obtained in study comparing Leishmania typing results in 16 European clinical laboratories, 2014
hsp70 sequencing
hsp70RFLP
Repetitive sequence
RFLP
ITS1 sequencing
kDNAMini-exon
sequencing
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Wrong complexWrong speciesCorrect subgenusCorrect complexCorrect species
ITS: internal transcribed spacer; hsp70: heat-shock protein 70 gene; kDNA: kinetoplast minicircle DNA; RFLP: restriction fragment length polymorphism.
a RFLP was performed on a fragment covering both ITS1 and ITS2 [14].
b One laboratory reported the use of two separate methods. Results E and M.
For each method, the number of correct typings to species, species complex, and subgenus level are shown in different colours. In addition, the incorrect species designations are indicated, some of which identified the wrong species in the correct complex (purple bars), others placing a strain in the wrong complex (red bars). The methods or combination of methods that were used to obtain the given results are shown on top.
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Figure 2Typing results for each of the 21 strains included in study comparing Leishmania typing results in 16 European clinical laboratories, 2014
L. donovani
L. a
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ajor
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L. (Viannia) subgenusL. (Leishmania) subgenus
Old World New World
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/JAP
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/M29
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/STI
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/85/
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/79/
M55
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Wrong complexWrong speciesCorrect subgenusCorrect complexCorrect species
MLSA: multilocus sequence analysis; WHO: World Health Organization.
a One laboratory reported the use of two separate methods.
b Strain MHOM/CO/88/UA316 is L. guyanensis based on MLEE, but L. panamensis based on MLSA (Table 1).
For each strain, the number of correct typings at species, species complex, and subgenus level are reported. In addition, the incorrect species designations are indicated, some of which identified the wrong species in the correct complex (purple bars), others placing an isolate in the wrong complex (red bars). The strain identification by WHO code (Table 1) is given with the abscissa. Species, complexes, and subgenera are represented on top, with an indication of the New or Old World strain origin.
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correct drug, route of administration (intralesional, oral systemic, or parenteral) and duration [7].
Unfortunately, for CL it is impossible to predict the species responsible for an ulcerating lesion clinically, and the morphology of amastigotes does not differ between species. When the geographical origin of infection is known, for instance when a patient in an endemic region is treated at a local hospital, the spe-cies can be guessed often from the known local epide-miology, as species distribution follows a geographical pattern [8]. However, especially in infectious disease clinics that treat patients who have stayed in various endemic countries, the geographic origin of infections may be unknown. For instance, people residing in Europe who have travelled outside Europe may come from, or have also visited, Leishmania-endemic areas within Europe, especially the Mediterranean basin. Even when the location of infection is known, sev-eral species can co-circulate in a given endemic area, in which case the species can only be determined by laboratory tests. Culture and subsequent isoenzyme analysis is time consuming and available in very few
specialised centres, so it is impractical as a front-line diagnostic test in clinical laboratories. Hence, well-per-formed reliable molecular methods are necessary for species identification.
Several Leishmania typing methods have been pub-lished (reviewed in [9]), and as a result each laboratory uses its own preferred assay. The most popular assays nowadays are those that can be applied directly to clin-ical samples, thereby circumventing the need for para-site isolation and culture. However, few tests have been standardised, and no commercial kits are currently available. As a result, clinical and epidemiological studies make use of various techniques, and in patient management other methods are often deployed. In this study we compare the typing performance in 16 clini-cal laboratories across Europe, which use a variety of methods for species discrimination.
Table 1Strains used, study comparing Leishmania typing results in 16 European clinical laboratories, 2014
Strain (WHO code) Culture name CNRLa Speciesb Reference typing methodc
MHOM/ET/83/130–83 LEM1118 Leishmania aethiopica MLEE, MLSAMHOM/GF/2002/LAV003 LEM4351 L. amazonensis MLEE, MLSAMHOM/VE/76/JAP78 LEM0391 L. amazonensis MLEE, MLSAMHOM/BR/75/M2903b LEM0396 L. braziliensis MLEE, MLSAMHOM/PE/83/STI139 LEM0781 L. braziliensis MLEE, MLSAMHOM/BO/2001/CUM555 NA L. braziliensis outlierd AFLP [12], WGS, MLSAMHOM/IN/--/LRC-L51 LEM1070 L. donovani MLEE, MLSAMHOM/KE/55/LRC-L53 LEM0707 L. donovani MLEE, MLSAMHOM/GF/86/LEM1034 LEM1034 L. guyanensis MLEE, MLSAMHOM/FR/78/LEM75 LEM0075 L. infantum MLEE, MLSAMCUN/BR/85/M9342 LEM2229 L. lainsoni MLEE, MLSAMHOM/IQ/86/CRE1 LEM0858 L. major MLEE, MLSAMHOM/BZ/82/BEL21 LEM0695 L. mexicana MLEE, MLSAMHOM/EC/87/EC103-CL8 LEM1554 L. mexicana MLEE, MLSAMDAS/BR/79/M5533 LEM2204 L. naiffi MLEE, MLSAMHOM/CO/86/UA126 LEM1047 L. panamensis MLEE, MLSAMHOM/CO/88/UA264 LEM1492 L. panamensis MLEE, MLSAMHOM/CO/88/UA316 LEM1505 L. panamensis / L. guyanensise MLEE, MLSAMHOM/PE/90/HB86 NA L. peruviana AFLP [12], WGS, MLSAMHOM/PE/90/LCA08 NA L. peruviana AFLP [12], WGS, MLSAMHOM/IL/80/SINGER LEM0617 L. tropica MLEE, MLSA
AFLP: amplified fragment length polymorphism; CNRL: Centre National de Référence des Leishmanioses (Montpellier, France); NA: not applicable; MLEE: multilocus enzyme electrophoresis; MLSA: multilocus sequence analysis; WGS: whole genome sequencing; WHO: World Health Organization.
a Identification in the Montpellier cryobank (Centre National de Référence des Leishmanioses).b For the taxonomic position of each species (subgenus and species complex), please refer to Figure 2.c Reference method used to determine the species of each isolate. MLEE [10]; MLSA based on seven genes [11]; AFLP analysis [12]; WGS
(unpublished results).d Group of distinct Leishmania braziliensis strains [9,12], also called L. braziliensis type 2 [15] or atypical L. braziliensis [18].e This strain was typed as L. panamensis by MLSA, and as L. guyanensis by MLEE.
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Methods
Participants and reference methodsTwenty one Leishmania isolates were typed by 16 labo-ratories in 12 countries in 2014. Table 1 lists the par-asite strains that were used in this study, along with the reference method for species identification. Strains identified with a Laboratoire d’Ecologie Médicale (LEM) code were provided by the Centre National de Référence des Leishmanioses in Montpellier, France, which assigns LEM codes to each cryopreserved cul-ture, while the remaining three strains were provided by the Institute of Tropical Medicine in Antwerp, Belgium.
Four highly informative reference methods were used: multilocus enzyme electrophoresis (MLEE [10]), multi-locus sequence analysis (MLSA [11], GenBank sequence accession numbers in Table 2), genome-wide amplified fragment length polymorphism (AFLP) analysis [12], and whole genome sequencing (unpublished results).
DNA was extracted from parasite cultures using either the DNeasy Blood and Tissue Kit or QIAamp DNA Mini Kit (Qiagen, www.qiagen.com), and the concentration was measured spectrophotometrically. The 21 DNAs were randomised at the United Kingdom (UK) National
External Quality Assessment Service for Parasitology (UKNEQAS, London, UK), and every study participant received a blind panel containing 50 µl of a 10 ng/µl DNA solution. The participating laboratories are listed in Table 3.
After performing the respective routine typing technol-ogy, each laboratory reported its results to UKNEQAS, who forwarded these along with the randomised code in one batch to the Institute of Tropical Medicine in Antwerp for analysis. Some participants used the term ‘L. braziliensis complex’ when referring to the L. (Viannia) subgenus, and where needed the reported results were adjusted. The results after these adjust-ments are presented in this analysis.
Genome targets for typingThe 16 laboratories used a total of five genome targets for typing (Table 4): the internal transcribed spacer 1 of the rDNA array (ITS1), the mini-exon, kinetoplast minicircle DNA (kDNA), the heat-shock protein 70 gene (hsp70), and a repetitive DNA sequence. One laboratory reported two sets of result from two different targets, which are treated in the analysis as if they were from separate laboratories, which is why the results section describes 17 instead of 16 outcomes. The targets were
Table 2GenBank sequence accession numbers from MLSA and hsp70, for sequences used in study comparing Leishmania typing results in 16 European clinical laboratories, 2014
WHO CODE LEMMLSA locus
hsp70 3,0980 4,0580 10,0560 12,0010 14,0130 31,0280 31,2610
MCUN/BR/85/M9342 2229 KT959002 KT959017 KT959032 KT959047 KT959062 KT959077 KT959092 LN907839MDAS/BR/79/M5533 2204 KT959001 KT959016 KT959031 KT959046 KT959061 KT959076 KT959091 FR872767MHOM/BO/2001/CUM555 NA KT959006 KT959021 KT959036 KT959051 KT959066 KT959081 KT959096 FR872760MHOM/BR/75/M2903b 396 KT958993 KT959008 KT959023 KT959038 KT959053 KT959068 KT959083 LN907832MHOM/BZ/82/BEL21 695 KT958994 KT959009 KT959024 KT959039 KT959054 KT959069 KT959084 LN907841MHOM/CO/86/UA126 1047 KT958997 KT959012 KT959027 KT959042 KT959057 KT959072 KT959087 LN907843MHOM/CO/88/UA264 1492 KT958998 KT959013 KT959028 KT959043 KT959058 KT959073 KT959088 LN907844MHOM/CO/88/UA316 1505 KT958999 KT959014 KT959029 KT959044 KT959059 KT959074 KT959089 LN907837MHOM/EC/87/EC103-CL8 1554 KT959000 KT959015 KT959030 KT959045 KT959060 KT959075 KT959090 LN907842MHOM/ET/83/130–83 1118 KC159315 KC159537 KC159093 KC159759 KC158871 KC159981 KC158649 LN907830MHOM/FR/78/LEM75 75 KC159255 KC159477 KC159033 KC159699 KC158811 KC159921 KC158589 LN907838MHOM/GF/2002/LAV003 4351 KT959003 KT959018 KT959033 KT959048 KT959063 KT959078 KT959093 LN907831MHOM/GF/86/LEM1034 1034 KT958996 KT959011 KT959026 KT959041 KT959056 KT959071 KT959086 LN907836MHOM/IL/80/SINGER 617 KC159287 KC159509 KC159065 KC159731 KC158843 KC159953 KC158621 LN907846MHOM/IN/--/LRC-L51 1070 KC159313 KC159535 KC159091 KC159757 KC158869 KC159979 KC158647 LN907834MHOM/IQ/86/CRE1 858 KC159299 KC159521 KC159077 KC159743 KC158855 KC159965 KC158633 LN907840MHOM/KE/55/LRC-L53 707 KC159294 KC159516 KC159072 KC159738 KC158850 KC159960 KC158628 LN907835MHOM/PE/1990/HB86 NA KT959004 KT959019 KT959034 KT959049 KT959064 KT959079 KT959094 LN907845MHOM/PE/1990/LCA08 NA KT959005 KT959020 KT959035 KT959050 KT959065 KT959080 KT959095 EU599089MHOM/PE/83/STI139 781 KT958995 KT959010 KT959025 KT959040 KT959055 KT959070 KT959085 LN907833MHOM/VE/76/JAP78 391 KT958992 KT959007 KT959022 KT959037 KT959052 KT959067 KT959082 EU599092
LEM: Laboratoire d’Ecologie Médicale; MLSA: multilocus sequence analysis; hsp70: heat-shock protein 70 gene; NA: not applicable; WHO: World Health Organization.
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analysed with PCR, generally followed by sequencing or restriction fragment length polymorphism (RFLP) analysis, as shown in Table 4 and Figure 1. Four labo-ratories used in-house sequencing, while five others used the service of an external sequencing facility. PCRs based on kDNA did not require post-PCR manipu-lations other than gel analysis.
Figure 1 indicates for each laboratory individually which method or methods were used, but not all samples were necessarily analysed with each method. Of the 16 laboratories, 11 used the ITS1 target, either applying RFLP (n=7) or sequencing (n=4). All of them based their analysis on the fragment described in [13], except for laboratory L which used a larger region also including ITS2 [14]. Five laboratories based typing on hsp70: four (A-D) used sequencing of the F fragment described in [15-17], while one (E) used the N fragment. Two labo-ratories (F and G) analysed this gene with RFLP [17,18]. Three laboratories used sequence analysis of the mini-exon gene: laboratory O [19,20], laboratory P [21], and laboratory Q [22]. Two laboratories based typing partly on kDNA: laboratory K [23], and laboratory L [24,25]. Finally, laboratory J complemented ITS1-RFLP with RFLP analysis of a repetitive DNA sequence [26].
Grading of resultsEach individual result was graded as follows. The best ranking was given to reported species agreeing with the reference methods, whereby L. garnhami was consid-ered a synonym of L. amazonensis [27]. Results report-ing MHOM/BO/2001/CUM555 as L. braziliensis were
considered correct. Although this strain belongs to a group of clearly distinguishable outliers (Table 1), it has so far not been described as a separate species. Next were identifications that reported the species complex rather than the actual species (see Figure 2), and were in agreement with the reference methods. The lowest ranking of correct results was given to those identify-ing the subgenus, i.e. L. (Viannia) or L. (Leishmania), without specification of species or species complex. Identification errors were graded at two levels. First, some laboratories reported a species within the cor-rect complex, but identified the wrong species within that complex. Second, some isolates were placed in an erroneous species complex altogether. A peculiar case was presented by strain MHOM/CO/88/UA316, which was L. guyanensis based on MLEE, but L. pan-amensis based on MLSA (Table 1). For this strain, all results reporting either L. guyanensis or L. panamensis were considered to have identified the correct species complex.
In a next level of the analysis, the cause of erroneous typings was sought by means of in-depth assessment of the methods. The reasons for different identifica-tion outcomes of laboratories using the same methods were also identified. Sequences from laboratories that based their typing on the same genes were compared by alignment in the software package MEGA5 [28].
ResultsResults from all analyses are summarised in Figure 1, details are available from [29]. One laboratory reported
Table 3Participants in study comparing Leishmania typing results in 16 European clinical laboratories, 2014
Institute City CountryInstitute of Tropical Medicine Antwerpa Antwerp BelgiumCentre National de Référence des Leishmaniosesa,b Montpellier FranceCentre Hospitalier Universitaire de Rennes Rennes FranceCharité-Universitätsmedizin Berlin Berlin GermanyHebrew University-Hadassah Medical Centre Jerusalem IsraelIstituto Superiore di Sanità Rome ItalyNational Institute for Infectious Diseases L. Spallanzani Rome ItalyInstituto de Higiene e Medicina Tropical Lisbon PortugalInstituto de Salud Carlos III Majadahonda SpainThe Public Health Agency of Sweden Stockholm SwedenSwiss Tropical and Public Health Institutea Basel SwitzerlandInstitute of Parasitology Zürich SwitzerlandAcademic Medical Center, University of Amsterdam Amsterdam The NetherlandsNational Institute for Public Health and the Environment Bilthoven The NetherlandsSt. Elisabeth Hospital Tilburg The NetherlandsEge University Medical School Izmir TurkeyHospital for Tropical Diseases London United Kingdom
Institutes are listed in alphabetical order based on country and city.a These laboratories provided parasite cultures and DNA.b This laboratory applied one of the reference methods (MLSA) and did not participate in the comparative study of typing outcomes.
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two sets of results because identification based on hsp70 sequences was sometimes in conflict with those of ITS1 sequencing. These results are listed separately from laboratories E and M respectively, which brings the number of reported result sets from the 16 labora-tories to 17. Most laboratories succeeded in typing all 21 samples, but in some cases results were reported for 20/21 isolates only (laboratories D, K, L, M). The total number of erroneous identifications amounted to 30, with 23 of these being classified in an incorrect species complex. On a total of 353 results, these rep-resent 8.5% and 6.5% respectively. The correct species was identified in 211 typing results (60%), while 58 (16%) identified the correct species complex, and 54 (15%) the correct subgenus. Eight laboratories made no incorrect assignments, while the laboratory with most errors (laboratory J) misidentified 10 out of 21 sam-ples, seven of which were placed in the wrong species complex. Laboratories relying only on kDNA and ITS1 more frequently reported results to the subgenus level, while laboratories using the mini-exon or hsp70 often succeeded in obtaining identification either to the spe-cies or complex level.
Figure 2 depicts the typing results for each strain, irre-spective of the methods used. The only two species that were correctly identified with all methods were L. tropica and L. major. Strains from the L. (Leishmania) subgenus were identified to either the species or com-plex level by all laboratories. This was in contrast to the 11 strains from the L. (Viannia) subgenus, each of which was typed by four to six laboratories only to the subgenus level. The error rate for both subgenera was comparable: 8.4% (14/167) for L. (Leishmania) and 8.6% (16/186) for L. (Viannia). The error rate in Old World strains was lower than for strains of the New World: 5.9% (6/102) and 9.6% (24/251) respectively.
When comparing the hsp70 sequences provided by four laboratories (A-D), there were marked differences in sequence quality. Three laboratories (A, B, C) suc-ceeded in sequencing the entire or nearly entire frag-ment F [17], with few or no sequence ambiguities. The sequence sets of two laboratories (A and C) contained one insertion and one deletion relative to the other data, indicating sequence mistakes as the gene shows no size variation [15,16]. In contrast, the quality of the fragment F sequences from one laboratory (D) was considerably lower. Sequences were largely incomplete at their 5’ end and to a lesser extent at their 3’ termi-nus, and numerous insertions, deletions, and unre-solved nucleotides (nt) were present. One laboratory (E) sequenced only the N fragment [17], but base call-ing quality was poor in the 40 terminal 3’ nt. The con-sensus hsp70 sequences were deposited in GenBank (Table 2).
Three laboratories (M, N, O) determined the ITS1 sequence of all isolates, while one laboratory (P) sequenced only MHOM/GF/2002/LAV003. The sequences of two laboratories (N and P) covered the entire amplified PCR product, while some of two others (O and M) were incomplete at the termini. Apart from some insertions in the sequences of one laboratory (N) and occasional unresolved nt in those of another (O), the sequences were identical, except for isolate MHOM/CO/88/UA316. Here, up to 9 nt differences were present in a 120 nt stretch.
Three laboratories (O, P, Q) determined the mini-exon sequences. For some strains the sequences of these laboratories were nearly identical, but for others large size differences of the determined fragment were seen, and deletions and nt identity discrepancies were observed. Also, many nt were not fully resolved.
DiscussionAs a general observation, eight laboratories who par-ticipated in this comparison typing performance made no errors, and often laboratories using the same typ-ing marker reported different results (Figure 1). Two of the ‘error-free’ laboratories obtained the highest typ-ing accuracy, with 20 out of 21 strains typed to the species level, and strain MHOM/CO/88/UA316 at the complex level. Using our reference methods MLSA and MLEE (Table 1), the latter species could not be classi-fied unequivocally, and hence results placing it in the L. guyanensis complex were regarded as correct. These two laboratories (A and B) based their typing on hsp70 gene sequencing, which was identified as one of the typing methods with the highest resolution in other comparative studies [9,15]. One other laboratory (C) also made use of this method, but typed several strains only to the complex level. Even though the hsp70 gene often permits distinction between closely related spe-cies, separating them is not always straight-forward. For instance, some MLEE-defined L. guyanensis have the same sequence as L. panamensis [16]. Because identifying the exact species within a given complex
Table 4Typing methods used in study comparing Leishmania typing results in 16 European clinical laboratories, 2014
Genomic locus / gene Analysis method Number of laboratoriesa
ITS1 RFLP [13,14] 7Sequencing [15] 4
hsp70 Sequencing [15,16] 5RFLP [17] 2
Mini-exon Sequencing [19-21] 3kDNA minicircles RFLP [24,25] 1
Specific PCR [23] 1Repetitive DNA RFLP [26] 1
ITS: internal transcribed spacer; hsp70: heat-shock protein 70 gene; kDNA: kinetoplast minicircle DNA; RFLP: restriction fragment length polymorphism.
a The total number is higher than the 16 participating laboratories, because several laboratories used different methods in parallel.
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can therefore be difficult, one laboratory (C) decided to identify the species complex rather than the exact species in case of doubt. Apparently the low sequence quality obtained by one of the participants (D) had no adverse effects on the results, probably because species-specific nt identities were not affected. The sequence quality was not influenced by the use of in-house vs external sequencing services.
One laboratory (E) reported four mistakes based on hsp70 sequences. As opposed to laboratories A-D, the analysis was based on a smaller part of the gene, frag-ment N [17], which is not suited for typing all species [15]. Nevertheless, several of these species were called based on a BLAST search in GenBank [https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch], from which the first listed species was regarded as the final result, regardless of identical similarity scores obtained from other species. In this process some spe-cies were by chance determined correctly, while others were erroneously identified. This stresses the impor-tance of correctly interpreting output lists generated by BLAST, because different species can have the same similarity score when the marker is too conservative for discriminating between them. To avoid such errors the species complex rather than the species itself should have been reported. On one occasion, the applied methodology even identified an erroneous complex, i.e. MHOM/ET/83/130–83 was typed as L. donovani instead of L. aethiopica, based on an erroneous anno-tation in GenBank. Indeed, several GenBank entries of [30] were wrongfully submitted as L. donovani, while they derived in fact from other species [16]. This illus-trates the importance of critically evaluating BLAST results, and underscores the importance of an agreed reference panel of sequences from trustworthy labo-ratories and knowledge of the limitations of a typing marker.
The same laboratory E reported a second results set based on ITS1 sequence analysis, listed under labora-tory M in Figure 1. Again, BLAST analysis was applied, and even though ITS1 is not suitable for discriminating L. braziliensis and L. guyanensis complex species [15], several species were reported. Except for one misclas-sified L. braziliensis outlier strain (Figure 2), species were correctly assigned by laboratory M. However, in several cases also other species showed the same sim-ilarity scores, and hence there was no ground for nam-ing the exact species. In contrast, another laboratory (N), which also used ITS1 sequence analysis, reported L. (Viannia) strains at subgenus level with no further attempt to determine the complex or species. Thereby they respected the limitations of ITS1, although some L. (Viannia) complexes could have been identified based on their data.
The majority of study participants that used ITS1 did not sequence the target, but relied on RFLP analy-sis. Laboratories basing their results on this method reported some typical errors: L. tropica was mixed up
with L. aethiopica; the L. donovani complex was con-fused with L. mexicana; unsuccessful attempts were made to separate L. infantum from L. donovani; and on one occasion L. amazonensis was identified as L. major. When digesting the PCR products with the popu-lar enzyme HaeIII, sufficient gel resolution is needed in order not to mix up the aforementioned species, as their RFLP fragments are similar in size. In addition, contrary to what was originally published [13], L. infan-tum cannot be distinguished from L. donovani [9] and therefore ITS1 can only type to the L. donovani com-plex, without further specification.
Two laboratories (F and G) complemented ITS-RFLP with hsp70-RFLP, and both mistook L. naiffi for L. brazilien-sis. This is a result of identical patterns generated from L. naiffi and many L. braziliensis strains with restriction endonucleases HaeIII and RsaI. The mistake could have been avoided by using the appropriate enzyme SduI [18].
Only one laboratory (J) made use of a repetitive DNA sequence originally described in [31]. In combina-tion with ITS1, 10 out of the 21 typings were incorrect, whereby seven strains were assigned to the wrong complex. Of the 10 mistakes, nine were made in the L. (Viannia) subgenus, while the remaining error was due to the unsuccessful separation of L. infantum from L. donovani. ITS1-RFLP is not suitable for discriminating these species, and the repetitive sequence RFLP was designed for typing Old World strains, where only the L. (Leishmania) subgenus is encountered. Such mis-takes once more underline the importance of knowing the limits of the typing marker chosen.
Kinetoplast DNA is primarily a useful marker to discrim-inate the two Leishmania subgenera, but is less suited for typing to the actual species level (reviewed in [9]). In combination with the fact that also ITS1-RFLP does not discriminate many L. (Viannia) species, the two lab-oratories (K and L) using these methods reported typ-ing mostly to the subgenus or species complex level. One of them (K) had a particularly high error rate (6/20) using these markers, probably related to the previously mentioned gel resolution problems and separation of L. infantum from L. donovani with ITS1-RFLP. In addition the laboratory used ‘L. braziliensis complex / L. guyan-ensis complex’ as a synonym for L. (Viannia), while two strains were L. naiffi and L. lainsoni.
With the mini-exon sequences, only two mistakes were reported. One laboratory (O) identified L. mexi-cana strain MHOM/EC/87/EC103-CL8 as L. donovani, but after disclosing the results realised a mistake in reporting, as their analysis actually did show the cor-rect species. In a comparative analysis of four markers [15], the mini-exon together with hsp70 were identified as the most discriminative markers worldwide, which is confirmed by the results presented here. Some species within the complexes can, however, not be resolved based on the mini-exon, as also reflected in the current
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analysis, where often complexes rather than species were identified.
When looking at the typing results for each of the 21 strains (Figure 2), it is apparent that strains of the L. (Viannia) subgenus were more often typed to the sub-genus level, while those of the L. (Leishmania) sub-genus were more often reported at the species level. Given that ITS1 was the most popular marker, this is a logical result in view of the poor discrimination of L. (Viannia) species by ITS1. Also the fact that for Old World strains 5.9% of typings were erroneous, in com-parison to 9.6% New World strains, relates to the use of methods that are tailored to Old World strains. Only two strains were identified to the species level by all laboratories and all methods: MHOM/IL/80/SINGER (L. tropica) and MHOM/IQ/86/CRE1 (L. major). The results show that several laboratories are currently unable to discriminate L. (Viannia) species, which is partly explained by the participation in the study of six groups that are situated in a European country where Leishmania is actively transmitted. Hence, they mainly diagnose patients infected by endemic species, and use methods primarily tailored to species in the Old World. On the contrary, the remaining laboratories are dealing only with imported leishmaniasis cases, which can originate from anywhere in the world, and for which the origin of infection is sometimes unknown. This forces them to apply assays that are able to iden-tify species from everywhere around the globe.
With regard to nomenclature, there is an evident need for standardisation. When the first results were reported, several laboratories used the term ‘L. bra-ziliensis complex’ to refer to L. (Viannia). For many years these have been synonyms, but current literature restricts this term to L. braziliensis and L. peruviana [27]. Another confusion can arise from the fact that each complex bears the name of one of its constituent species. For instance, a typing outcome reported as ‘L. guyanensis’ has to be clearly distinguished from ‘L. guyanensis complex’. Even though this particular prob-lem did not seem to occur in our analysis, one could easily envision such occurrence. One laboratory (K) reported several results as ‘L. braziliensis complex / L. guyanensis complex’ for referring to L. (Viannia), but with this term L. naiffi and L. lainsoni were excluded.
Finally, the particular case of strain MHOM/CO/88/UA316 draws attention to problems in species defini-tions, as this strain was typed as L. guyanensis with MLEE, but as L. panamensis with MLSA (Table 1). Reported correct results for this strain were either L. guyanensis complex, L. guyanensis, or L. panamensis, but this was irrespective of the method or target used [29]. Such occasional dubious results are unavoidable when dealing with closely related species, in particular L. guyanensis-L. panamensis; L. braziliensis-L. peruvi-ana; L. mexicana-L. amazonensis; and L. donovani-L. infantum [9]. Also newly documented parasite species such as L. martiniquensis [32] and L. waltoni [33], and
variants as the L. braziliensis outlier [9,12,15,18] fur-ther complicate the interpretation of typing results. It is therefore of utmost importance that species identi-fication is performed with a well-documented standard operating procedure (SOP), clearly describing not only experimental procedures, but also in detail how results should be analysed, interpreted, and reported.
The current study was performed on cultured parasite isolates, so all participants received a high amount of pure parasite DNA. Yet, 8.5% errors were seen, and in four cases no result was obtained. When dealing with patient material, the amount of parasite DNA is much lower, and vastly exceeded by human DNA. As the cur-rent study did not assess the sensitivity of the meth-ods used, it is expected that typing success based on clinical samples will be considerably lower. In view of the fact that only recognised reference laboratories participated in this study, there is a clear need for opti-misation. On the other hand, in many clinical settings the suspected origin of infection can help in interpreta-tion of typing outcomes, thereby possibly lowering the error rate.
ConclusionsThere is considerable room for improvement of cur-rent Leishmania typing strategies, and inter-labora-tory comparisons such as the one we conducted can contribute to enhance typing quality. Whichever the clinical need for determining the subgenus, complex, or species, and whichever the technology used in a particular setting, typing should be based on a well-defined and validated SOP designed by an expert in Leishmania taxonomy. This SOP should cover not only testing, but also analysis and interpretation proce-dures, and a clear description of how species should be named and reported, taking into account the limita-tions of each marker and technique, and the problem of resolving closely related species or occasional inter-species hybrids. Validation should be performed on a sufficient amount of reference isolates from various geographic origins to cover each species’ variability. When using sequencing, sequence errors should be avoided, and a well-validated sequence reference set is recommended over BLAST analysis using GenBank, which lacks quality control. In cases where treatment is species- or complex-dependent, clinicians should be made aware of the limitations of the technology used whenever results are reported, especially when closely related species are involved. The use of real-time PCR assays developed for specific complexes or species could speed up typing and facilitate interpretation of results, but currently no globally applicable methods are available. As previously recommended [15] and also apparent from this analysis, hsp70 and the mini-exon currently offer the best Leishmania typing tools world-wide, and the use of ITS1 should be restricted to the Old World. Setting up similar evaluations out-side Europe, in institutes in endemic as well as non-endemic countries, would shed additional light on the quality of Leishmania typing across the globe.
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AcknowledgementsThe authors would like to thank the ESCMID study group for Clinical Parasitology (ESGCP, headed by Titia Kortbeek, National Institute for Public Health and the Environment, RIVM, Bilthoven, The Netherlands) for financial support (ESCMID Study Group Research Grant 2013 to Peter L. Chiodini). We are grateful to Jean-Pierre Gangneux (ESGCP co-ordinator for leishmaniasis, Centre Hospitalier Universitaire de Rennes, Rennes, France) and the LeishMan consortium [www.leishman.eu] for conceptual support and promoting the study. We acknowledge the valuable comments of Titia Kortbeek, and the technical assistance of Sofia Andersson (The Public Health Agency of Sweden, Stockholm, Sweden), José M. Cristóvão (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, UNL, Lisbon, Portugal), Mehmet Karakus (Ege University, Faculty of Medicine, Department of Parasitology, Izmir, Turkey), Ilse Maes (Institute of Tropical Medicine, Antwerp, Belgium), Abed Nasereddin (Hebrew University, Hadassah Medical Centre, Jerusalem, Israel), Chris Stalder (Swiss Tropical and Public Health Institute, Basel, Switzerland), Antonietta Toffoletti and Antonella Vulcano (National Institute for Infectious Diseases (INMI) Lazzaro Spallanzani, Rome, Italy), Carla Wassenaar (Academic Medical Center, Amsterdam, The Netherlands), Julie Watson and Spencer Polley (Hospital for Tropical Diseases, London, United Kingdom). Gert Van der Auwera is supported by the Third Framework pro-gram between ITM and the Belgian Directorate General for Development. Peter L. Chiodini is supported by the Biomedical Research Centre of the University College London Hospitals and National Institute for Health Research.
Conflict of interestNone declared.
Authors’ contributionsGert Van der Auwera, Aldert Bart, Ingrid Felger, Christophe Ravel, Jean-Claude Dujardin, and Peter L. Chiodini conceptu-alised the study. Gert Van der Auwera coordinated the study, analysed the data, and drafted the publication. Christophe Ravel and Gert Van der Auwera provided the cultures, from which DNA was extracted by Ingrid Felger and Gert Van der Auwera. Monika Manser was responsible for blinding the sam-ples and collecting the results. Gert Van der Auwera, Aldert Bart, Carmen Chicharro, Sofia Cortes, Leigh Davidsson, Trentina Di Muccio, Ingrid Felger, Maria Grazia Paglia, Felix Grimm, Gundel Harms, Charles L. Jaffe, Christophe Ravel, Florence Robert-Gangneux, Jeroen Roelfsema, Seray Töz, and Jaco J. Verweij supervised or carried out the assays. All authors gave their input on the manuscript draft.
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License and copyrightThis is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi-cate if changes were made.
This article is copyright of the authors, 2016.
20 www.eurosurveillance.org
Letter
Community-wide outbreaks of haemolytic uraemic syndrome associated with Shiga toxin-producing Escherichia coli O26 in Italy and Romania: a new challenge for the European Union
E Severi ¹ , F Vial ¹ , E Peron 2 3 , O Mardh ¹ , T Niskanen ¹ , J Takkinen 1 1. European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden2. European Programme for Intervention Epidemiology Training (EPIET), European Centre for Disease Prevention and Control
(ECDC), Stockholm, Sweden3. Gastrointestinal, zoonosis and tropical diseases unit, Department of infectious diseases epidemiology, Robert Koch Institute,
Berlin, GermanyCorrespondence: Ettore Severi ([email protected])
Citation style for this article: Severi E, Vial F, Peron E, Mardh O, Niskanen T, Takkinen J. Community-wide outbreaks of haemolytic uraemic syndrome associated with Shiga toxin-producing Escherichia coli O26 in Italy and Romania: a new challenge for the European Union. Euro Surveill. 2016;21(49):pii=30420. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2016.21.49.30420
Article submitted on 02 December 2016 / accepted on 08 December 2016 / published on 08 December 2016
To the editor: In their recent article in Eurosurveillance, Germinario et al. describe a community-wide outbreak of Shiga toxin 2-producing Escherichia coli (STEC) O26:H11 infections associated with haemolytic uraemic syndrome (HUS) and involving 20 children between 11 and 78 months of age in southern Italy during the sum-mer 2013 [1]. The investigation identified an associa-tion between STEC infection and consumption of dairy products from two local milk-processing establish-ments. We underline striking similarities to a recent multi-country STEC O26 outbreak in Romania and Italy and discuss the challenges that STEC infections and their surveillance pose at the European level.
In March 2016, Peron et al. published, also in Eurosurveillance, early findings of the investigation of a community-wide STEC infection outbreak in south-ern Romania [2]. As at 29 February 2016, 15 HUS cases with onset of symptoms after 24 January 2016, all but one in children less than two years of age, had been identified, three of whom had died. Aetiological con-firmation was retrospectively performed through sero-logical diagnosis and six cases were confirmed with STEC O26 infection. Shortly after this publication, and following the identification of the first epidemiologi-cally-linked case in central Italy, the European Centre for Disease Prevention and Control (ECDC) and the European Food Safety Authority (EFSA) published a joint Rapid Outbreak Assessment [3]. The Italian and Romanian epidemiological, microbiological and envi-ronmental investigations implicated products from a milk-processing establishment in southern Romania as a possible source of infection. The dairy plant exported milk products to at least four European Union (EU)
countries. The plant was closed in March 2016 and the implicated food products recalled or withdrawn from the retail market.
Pulsed Field Gel Electrophoresis (PFGE) and whole genome sequencing (WGS) analyses did not estab-lish a microbiological link between the Italian (2013) and the Romanian/Italian (2016) outbreaks (personal communication, Stefano Morabito, October 2016). However, the epidemiological similarities between the two community-wide outbreaks associated with HUS and STEC O26 infections, mostly affecting young chil-dren and implicating dairy products, are notable. While raw milk and unpasteurised dairy products are well known potential sources of STEC infection, milk prod-ucts, as highlighted by Germinaro et al. [1], have been rarely implicated in community-wide STEC outbreaks in the past, emphasising an emerging risk of STEC O26 infection associated with milk products.
Reporting of STEC O26 infections has been steadily increasing in the EU since 2007, partly due to improved diagnostics of non-O157 sero-pathotypes [4]. The attention to non-O157 STEC sero-pathotypes rose con-siderably after the severe STEC O104 outbreak that took place in Germany and France in 2011 during which almost 4,000 cases and more than 50 deaths were reported [5]. In light of the recently published out-breaks related to dairy products and the simultaneous increased reporting of isolations of STEC O26 from milk and milk products in the EU/European Economic Area (EEA) [6], strengthening STEC surveillance in humans and food and enhancing HUS surveillance in children less than five years of age is warranted. Paediatric
21www.eurosurveillance.org
nephrologists should be sensitised to this effect and further joint studies between food and public health sectors be increased.
Conflict of interestNone declared.
Authors’ contributionsES drafted the letter to the Editor. All authors reviewed, com-mented and accepted its final version.
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MS, Tafuri S, et al. , all participants of the Outbreak investigation team. Community-wide outbreak of haemolytic uraemic syndrome associated with Shiga toxin 2-producing Escherichia coli O26:H11 in southern Italy, summer 2013.Euro Surveill. 2016;21(38):30343. DOI: 10.2807/1560-7917.ES.2016.21.38.30343 PMID: 27684204
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License and copyrightThis is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi-cate if changes were made.
This article is copyright of the authors, 2016.
22 www.eurosurveillance.org
News
New version of the Epidemic Intelligence Information System for food- and waterborne diseases and zoonoses (EPIS-FWD) launched
CM Gossner ¹ 1. European Centre for Disease Prevention and Control (ECDC), Stockholm, SwedenCorrespondence: Celine Gossner ([email protected])
Citation style for this article: Gossner CM. New version of the Epidemic Intelligence Information System for food- and waterborne diseases and zoonoses (EPIS-FWD) launched. Euro Surveill. 2016;21(49):pii=30422. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2016.21.49.30422
Article published on 08 December 2016
On 1 December 2016 the third version of the Epidemic Intelligence Information System for food- and water-borne diseases and zoonoses (EPIS-FWD) was launched. With this development, the European Centre for Disease Prevention and Control (ECDC) moved one step further towards the One Health approach.
In collaboration with the European Food Safety Authority (EFSA), the Molecular Typing Cluster Investigation (MTCI) module was expanded to also allow the assess-ment of Salmonella, Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes microbiological clusters based on non-human isolates (i.e. food, feed, animal and environmental) and on a mix of non-human and human isolates.
Depending on the type of cluster assessed, the MTCIs are coordinated by ECDC or EFSA or jointly by both agencies together with public health and/or food safety and veterinary experts from the involved European Union (EU) and European Economic Area (EEA) Member States.
ECDC collects human typing data through the European Surveillance System (TESSy) since 2013 [1]. Typing data from non-human isolates can now be submitted by the food and veterinary authorities of the EU/EEA Member States through the EFSA molecular typing data collec-tion system. Furthermore, the joint ECDC-EFSA molecu-lar typing database allows the comparison of the typing data collected by ECDC and EFSA.
First launched in March 2010, the Epidemic Intelligence Information System for food- and waterborne diseases and zoonoses (EPIS-FWD) has become an important tool for assessing on-going public health risks related to FWD events worldwide. Currently, 52 countries from five continents have access to the outbreak alerts in the EPIS-FWD [2].
Since its launch, 305 outbreak alerts have been assessed through the EPIS-FWD; 32 (10%) were from countries outside of the EU/EEA which underlines the global dimension of the system.
The Health Security Committee, a part of the European Commission and the officially nominated public health risk management authority in the EU/EEA, has access to the EPIS-FWD to ensure the link between risk assessment and risk management. The World Health Organisation (WHO), including the International Network of Food Safety Authorities (INFOSAN) man-aged jointly by the Food and Agriculture Organisation of the United Nations (FAO) and WHO, is invited to con-tribute to the discussions in the EPIS-FWD when inter-national outbreaks involve non-EU/EEA countries.
Through this new version of EPIS-FWD, ECDC and EFSA are encouraging the sharing of data between sectors and aspire to strengthen the multi-sectorial collabora-tion at international and national levels.
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Information Sources 2016 [cited 2016 28 Nov]; Available from: http://ecdc.europa.eu/en/aboutus/what-we-do/epidemic-intelligence/Pages/EpidemicIntelligence_Tools.aspx.
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